STANDARD OPERATING PROCEDURE (SOP): Rh (D) Typing

BLOOD CENTRE NAME

SCOPE AND APPLICATION:

This Standard Operating Procedure provides the method to be followed to determine the Rh D type of an individual and ensure the reliability of the result. This procedure describes the method for detection of Rh D antigen on the red cells.

RESPONSIBILITY :

It is the responsibility of Medical Technologist (Lab) in the serology area to perform the Rh D typing of an individual using one monoclonal & one blended reagent. If a discrepancy is encountered between the two batches of anti-D, the test should be repeated and confirmed by Indirect Coombs’ Test. It is the responsibility of all staff performing the Rh D typing to ensure that quality controlled reagents and proper cell concentration are used.

 REFERENCES :

• Model standard operating procedures for blood transfusion service, WHO, New Delhi 2002 
• Technical Manual of American Association of Blood Banks, 18^th edition 

MATERIAL REQUIRED: 

            Equipment: 

• Refrigerator to store samples and reagents at 2- 6◦ C 
• Table top centrifuge 
• Microscope 

            Specimen: 

• Blood samples of donors/ patients 

            Reagents: 

• Anti-D Blended 
• Anti-D Monoclonal 
• Rh control reagents (positive & negative) 
• 0.9 % normal saline 

           Glassware: 

• Test tubes 
• Plastic pipette 
• Glass slides 

           Miscellaneous: 

• Disposal box 
• Glass beakers – 2 
• plastic racks to hold test tubes

 PROCEDURE: 

PRINCIPLE: 

Testing with anti-D is necessary to determine if red blood cells possess or lack Rh D blood group antigen. Presence of agglutination is a positive test result, which indicates the presence of the D antigen on the red blood cells. 

 PROCEDURE FOR  RH (D) TYPING: 

• Label tubes with patient/unit and test identification
• Add 2 drop of reagent anti-D to the test tube.
• Add 1 drop of the 5% cell suspension to each test tube.
• Mix well and centrifuge at 1000 rpm for 1min (incubation temperature and time depends on manufacturer’s instructions).
• Gently re-suspend the cell button and examine macroscopically for agglutination
• And records the results immediately

PROCEDURE FOR DETERMINATION OF WEAK D :

Performed in all the sam[ples giving negative reaction for Rh(D) antigen on Rh typing.

PROCEDURE :

• Label 1 test tube with patient name/number
• Add 2 drops of preferably polyclonal anti-D(IgG) or blended monoclonal IgG and IgM
• Add 1 drop of 5% Rh –D negative red cells
• Incubate at 37o C X 45-60 mins
• Wash the incubated tube 3 times with normal saline and decant the last wash completely
• Add 1 drop of  polyspecific AHG to the washed red cell button
• Centrifuge at 1000rpm for I min
• Gently re-suspend the cell button and examine macroscopically for agglutination
• And records the results immediately
• Add 1 drop of check cells to the tube giving negative agglutination after adding result
• Centrifuge at 1000rpm for I min
• Record the result

INTERPRETATION :

DOCUMENTATION:

• Enter the results of donor grouping in the donor grouping register and computer.
•  Enter the results of patients grouping in the patient grouping register, blood group requisition form, serial case number register and computer.